Male reproductive traits are differentially affected by dietary macronutrient balance but unrelated to adiposity

Dietary factors influence male reproductive function in both experimental and epidemiological studies. However, there are currently no specific dietary guidelines for male preconception health. Here, we use the Nutritional Geometry framework to examine the effects of dietary macronutrient balance on reproductive traits in C57BL/6 J male mice. Dietary effects are observed in a range of morphological, testicular and spermatozoa traits, although the relative influence of protein, fat, carbohydrate, and their interactions differ depending on the trait being examined. Interestingly, dietary fat has a positive influence on sperm motility and antioxidant capacity, differing to typical high fat diet studies where calorie content is not controlled for. Moreover, body adiposity is not significantly correlated with any of the reproductive traits measured in this study. These results demonstrate the importance of macronutrient balance and calorie intake on reproductive function and support the need to develop specific, targeted, preconception dietary guidelines for males.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Two studies in flies have used the NGF to assess paternal effects, the 1st using n=15 with 20 diets (Functional Ecology 2016. 30:1675-1686, and the 2nd using n=20 with 15 diets (Proceedings B 2017. 284:20171492). To be able to detect comparable effect sizes, similar sample sizes were required for the mouse study, but were more limited by ethical considerations compared to invertebrate work.
No data exclusion.
Following an acclimation period, each cage of 3x males was randomly assigned to a diet treatment by one operator.
Analysis of testicular architecture (Johnsen scoring) was performed by a blinded observer (i.e. blinded to group allocation during data collection), with samples prepared by another team member. For the two other subjective measures (sperm motility, sperm morphology), the same observer was responsible for preparing and assessing samples and so could not be blinded to group allocation. All other measures used were not subjective, thus blinding was not used.
Immediately following sacrifice, sperm were isolated. Both cauda epididymides were isolated and transferred to a Petri dish containing warmed 600µL Ham's F10 medium (with 25mM HEPES and 1mM L-glutamine, catalogue number 12390-035, Thermo Fisher Scientific, Riverstone, Australia) supplemented with 1mg/mL polyvinyl alcohol (87-90% hydrolyzed, average molecular weight 30,000 -70,000), referred to subsequently as Ham's F10 + PVA. Epididymides were cut 5 times with a sterile scalpel blade and the tissue incubated for 15 min at 37°C. Tissues were discarded and concentration of the isolated spermatozoa was determined using a Neubauer haemocytometer. Samples were subsequently diluted to 20 x 106 spermatozoa/mL with Ham's F10 + PVA and maintained at 37°C. Viability was assessed using the LIVE/DEAD fixable red dead cell stain (Thermo Fisher Scientific) as per the manufacturer's directions. Briefly, a 50µL aliquot of samples diluted to 20x106 spermatozoa/mL as described above was further diluted to 2x106spermatozoa/mL with phosphate buffered saline (PBS) supplemented with 1mg/mL polyvinyl alcohol, referred to subsequently as PBS + PVA. One microlitre of stain was added and samples were incubated at 37°C for 30 min, then washed with 500µL PBS + PVA (600xg, 2 min). The resultant pellet was fixed with 10% neutral buffered formalin for 15 min, washed with 1mL PBS + PVA (600xg, 2 min) and stored at 4ºC in the dark until assessment (up to 4 days later). Reactive oxygen species production was assessed using 2",7"-dichlorofluorescein diacetate (H2DCFDA). A 100µL aliquot of samples diluted to 20x106 spermatozoa/mL as described above was stained (final concentration 5µM) at 37°C for 30 min. Excess stain was removed by centrifuging samples (600xg, 2 min) and resuspending in fresh Ham's F10 + PVA. Samples were assessed for baseline ROS production immediately following stain loading and after a further 1 h incubation period. At each time point, an aliquot was counterstained with propidium iodide (PI; final concentration 6µM) for 5 min prior to assessment to discriminate the viable population.
Becton Dickinson LSRFortessa X-20 FACSDiva (v 9.0), FlowJo software (v 10.7.2) Sperm accounted for~50-90% of total events (based on forward vs side scatter), single cells accounted for >80% of the sperm population (based on FSC area vs FSC height). Identification of sperm was checked by back gating stained cells to original population.
All samples were gated firstly on the basis of forward and side scatter to isolate spermatozoa from debris, and subsequently on the basis of forward scatter area and height to isolate single cells. For the fixable viability stain, a 610/10 BP histogram was